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Glycerol restores heat-induced p53-dependent apoptosis of human glioblastoma cells bearing mutant p53.

Ohnishi T, Ohnishi K, Takahashi A - BMC Biotechnol. (2002)

Bottom Line: The phosphorylation of mp53 at serine15 was suppressed by an inhibitor of the phosphatidylinositol 3-kinase (PI3-K) family.These results suggest that glycerol is effective in inducing conformational change of phosphorylated p53 and restoring mp53 to wtp53 function, leading to enhanced heat sensitivity through the induction of apoptosis.This novel tool for enhancement of heat sensitivity in cancer cells bearing mp53 may be applicable for p53-targeted hyperthermia, because mutation or inactivation of p53 is observed in approximately 50% of human cancers.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521, Japan. tohnishi@nmu-gw.naramed-u.ac.jp

ABSTRACT

Background: We have previously reported that glycerol acts as a chemical chaperone to restore the expression of WAF1 in some human cancer cell lines bearing mutant p53. Since the expression of WAF1 is up-regulated by activated wildtype p53, glycerol appears to restore wtp53 function. The aim of the present study is to examine the restoration of heat-induced p53-dependent apoptosis by glycerol in human glioblastoma cells (A-172) transfected with a vector carrying a mutant p53 gene (A-172/mp53 cells) or neo control vector (A-172/neo cells).

Results: A-172/mp53 cells showed heat resistance compared with A-172/neo cells but A-172/mp53 cells in turn became heat sensitive when pre-treated with glycerol before heat treatment. The accumulation of Bax in the A-172/mp53 cells was induced by heating with glycerol pre-treatment, but not without it, whereas the accumulation in the A-172/neo cells was induced in both cases. Furthermore, mp53 extracted from heated cells came to bind to the sequence specific region after heating combined with glycerol pre-treatment. The phosphorylation of mp53 at serine15 was suppressed by an inhibitor of the phosphatidylinositol 3-kinase (PI3-K) family.

Conclusion: These results suggest that glycerol is effective in inducing conformational change of phosphorylated p53 and restoring mp53 to wtp53 function, leading to enhanced heat sensitivity through the induction of apoptosis. This novel tool for enhancement of heat sensitivity in cancer cells bearing mp53 may be applicable for p53-targeted hyperthermia, because mutation or inactivation of p53 is observed in approximately 50% of human cancers.

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Gel mobility-shift assay of nuclear extracts or whole cell extracts from A-172/neo or A-172/mp53/248 cells to p53CON. in vivo, the cells were treated with glycerol (0.6 M), wortmannin (20 μM) and heat (at 44°C for 30 min). The nuclear fraction was extracted from the treated cells 6 h after heating. in vitro, the whole cell extracts prepared from intact cells, and then they were treated with glycerol, wortmannin and heat.
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Figure 3: Gel mobility-shift assay of nuclear extracts or whole cell extracts from A-172/neo or A-172/mp53/248 cells to p53CON. in vivo, the cells were treated with glycerol (0.6 M), wortmannin (20 μM) and heat (at 44°C for 30 min). The nuclear fraction was extracted from the treated cells 6 h after heating. in vitro, the whole cell extracts prepared from intact cells, and then they were treated with glycerol, wortmannin and heat.

Mentions: Furthermore, to confirm the conformational change of mp53 to wtp53 suggested by Western blot analysis, the DNA binding activity of p53 for p53CON was measured in nuclear proteins extracted from A-172/neo or A-172/mp53/248 cells using the gel mobility-shift assay. It is known that wtp53 can bind to p53CON homologous to a specific DNA sequence located upstream of the bax gene which positively controls apoptosis [24]. In agreement of this, the binding activity of wtp53 significantly increased in A-172/neo cells treated with heat (44°C, 30 min) or combination of heat and glycerol (0.6 M) (Fig. 3, the upper column, lane 3 and 4). In contrast, a slight increase in the DNA binding activity of the nuclear proteins was observed when A-172/mp53/248 cells were treated with heat (44°C, 30 min) (Fig. 3, the lower column, lanes 3). The increase may be due to endogenous wtp53 in A-172/mp53/248 cells. The defective DNA binding ability of p53 from heat-treated A-172/mp53/248 cells may be due to the dominant negative nature of mp53 protein [16,17]. It is known that mp53 has no ability to induce apoptosis, because most mp53 can not bind to a specific DNA sequence [5,6]. On the other hand, when A-172/mp53/248 cells were heated in the presence of glycerol (0.6 M) before heating, they showed clear increased DNA binding activity of p53 to p53CON (Fig. 3, the lower column, lane 4). This result shows that mp53 underwent conformational change to wtp53. The enhanced heat sensitivity observed in glycerol-pretreated A-172/mp53/143 cells (Fig. 1a) might be induced through bax gene expression up-regulated by the activated mp53. No binding activity of p53 was observed when whole cell proteins extracted from intact A-172/neo or A-172/mp53/248 cells were treated with heat, heat plus glycerol or heat plus glycerol plus wortmannin (Fig. 3, both columns, lanes 8, 9 and 10). Thus, the acquisition of binding activity of wtp53 or glycerol-treated mp53 is likely to demand cellular signal transduction as described above. Furthermore, the increased DNA binding activity of nuclear proteins from A-172/neo or A-172/mp53/248 cell was suppressed by wortmannin (Fig. 3, lane 5). This suggests that the PI3-K family mediates glycerol-induced restoration of the DNA binding ability of wtp53 and mp53 after heating. On Western blot analysis (Fig. 2), phosphorylation of serine 15 seems to be required for this process.


Glycerol restores heat-induced p53-dependent apoptosis of human glioblastoma cells bearing mutant p53.

Ohnishi T, Ohnishi K, Takahashi A - BMC Biotechnol. (2002)

Gel mobility-shift assay of nuclear extracts or whole cell extracts from A-172/neo or A-172/mp53/248 cells to p53CON. in vivo, the cells were treated with glycerol (0.6 M), wortmannin (20 μM) and heat (at 44°C for 30 min). The nuclear fraction was extracted from the treated cells 6 h after heating. in vitro, the whole cell extracts prepared from intact cells, and then they were treated with glycerol, wortmannin and heat.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC111188&req=5

Figure 3: Gel mobility-shift assay of nuclear extracts or whole cell extracts from A-172/neo or A-172/mp53/248 cells to p53CON. in vivo, the cells were treated with glycerol (0.6 M), wortmannin (20 μM) and heat (at 44°C for 30 min). The nuclear fraction was extracted from the treated cells 6 h after heating. in vitro, the whole cell extracts prepared from intact cells, and then they were treated with glycerol, wortmannin and heat.
Mentions: Furthermore, to confirm the conformational change of mp53 to wtp53 suggested by Western blot analysis, the DNA binding activity of p53 for p53CON was measured in nuclear proteins extracted from A-172/neo or A-172/mp53/248 cells using the gel mobility-shift assay. It is known that wtp53 can bind to p53CON homologous to a specific DNA sequence located upstream of the bax gene which positively controls apoptosis [24]. In agreement of this, the binding activity of wtp53 significantly increased in A-172/neo cells treated with heat (44°C, 30 min) or combination of heat and glycerol (0.6 M) (Fig. 3, the upper column, lane 3 and 4). In contrast, a slight increase in the DNA binding activity of the nuclear proteins was observed when A-172/mp53/248 cells were treated with heat (44°C, 30 min) (Fig. 3, the lower column, lanes 3). The increase may be due to endogenous wtp53 in A-172/mp53/248 cells. The defective DNA binding ability of p53 from heat-treated A-172/mp53/248 cells may be due to the dominant negative nature of mp53 protein [16,17]. It is known that mp53 has no ability to induce apoptosis, because most mp53 can not bind to a specific DNA sequence [5,6]. On the other hand, when A-172/mp53/248 cells were heated in the presence of glycerol (0.6 M) before heating, they showed clear increased DNA binding activity of p53 to p53CON (Fig. 3, the lower column, lane 4). This result shows that mp53 underwent conformational change to wtp53. The enhanced heat sensitivity observed in glycerol-pretreated A-172/mp53/143 cells (Fig. 1a) might be induced through bax gene expression up-regulated by the activated mp53. No binding activity of p53 was observed when whole cell proteins extracted from intact A-172/neo or A-172/mp53/248 cells were treated with heat, heat plus glycerol or heat plus glycerol plus wortmannin (Fig. 3, both columns, lanes 8, 9 and 10). Thus, the acquisition of binding activity of wtp53 or glycerol-treated mp53 is likely to demand cellular signal transduction as described above. Furthermore, the increased DNA binding activity of nuclear proteins from A-172/neo or A-172/mp53/248 cell was suppressed by wortmannin (Fig. 3, lane 5). This suggests that the PI3-K family mediates glycerol-induced restoration of the DNA binding ability of wtp53 and mp53 after heating. On Western blot analysis (Fig. 2), phosphorylation of serine 15 seems to be required for this process.

Bottom Line: The phosphorylation of mp53 at serine15 was suppressed by an inhibitor of the phosphatidylinositol 3-kinase (PI3-K) family.These results suggest that glycerol is effective in inducing conformational change of phosphorylated p53 and restoring mp53 to wtp53 function, leading to enhanced heat sensitivity through the induction of apoptosis.This novel tool for enhancement of heat sensitivity in cancer cells bearing mp53 may be applicable for p53-targeted hyperthermia, because mutation or inactivation of p53 is observed in approximately 50% of human cancers.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521, Japan. tohnishi@nmu-gw.naramed-u.ac.jp

ABSTRACT

Background: We have previously reported that glycerol acts as a chemical chaperone to restore the expression of WAF1 in some human cancer cell lines bearing mutant p53. Since the expression of WAF1 is up-regulated by activated wildtype p53, glycerol appears to restore wtp53 function. The aim of the present study is to examine the restoration of heat-induced p53-dependent apoptosis by glycerol in human glioblastoma cells (A-172) transfected with a vector carrying a mutant p53 gene (A-172/mp53 cells) or neo control vector (A-172/neo cells).

Results: A-172/mp53 cells showed heat resistance compared with A-172/neo cells but A-172/mp53 cells in turn became heat sensitive when pre-treated with glycerol before heat treatment. The accumulation of Bax in the A-172/mp53 cells was induced by heating with glycerol pre-treatment, but not without it, whereas the accumulation in the A-172/neo cells was induced in both cases. Furthermore, mp53 extracted from heated cells came to bind to the sequence specific region after heating combined with glycerol pre-treatment. The phosphorylation of mp53 at serine15 was suppressed by an inhibitor of the phosphatidylinositol 3-kinase (PI3-K) family.

Conclusion: These results suggest that glycerol is effective in inducing conformational change of phosphorylated p53 and restoring mp53 to wtp53 function, leading to enhanced heat sensitivity through the induction of apoptosis. This novel tool for enhancement of heat sensitivity in cancer cells bearing mp53 may be applicable for p53-targeted hyperthermia, because mutation or inactivation of p53 is observed in approximately 50% of human cancers.

Show MeSH
Related in: MedlinePlus